Mesenchymal Stem Cells Expressing TNF-alpha Receptors

ABSTRACT

Mesenchymal stem cells which express TNF-α receptor Type I in an amount of at least 13 pg/10 6  cells. Such mesenchymal stem cells inhibit the proliferation of lymphocytes and may be employed, in particular, in the treatment of graft-versus-host disease.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/228,327, filed on Mar. 28, 2014, which is a continuation of U.S. patent application Ser. No. 13/906,592, filed on May 31, 2013, which is a divisional of U.S. patent application Ser. No. 13/402,498, filed on Feb. 22, 2012 (now U.S. Pat. No. 8,486,695), which is a continuation of U.S. patent application Ser. No. 12/091,391, filed on Sep. 15, 2008, now abandoned, which is a National Stage Entry of international patent application number PCT/US07/00274, filed on Jan. 5, 2007, which claims priority to U.S. Provisional Patent Application Ser. No. 60/759,157, filed on Jan. 13, 2006, now expired; the contents of all of which are hereby incorporated by reference in their entireties.

BRIEF SUMMARY OF THE INVENTION

This invention relates to mesenchymal stem cells. More particularly, this invention relates to mesenchymal stem cells which express tumor necrosis factor-alpha (TNF-α) receptors, and in particular, the tumor necrosis factor-alpha (TNF-α) receptor Type I (TNFRI), in an amount of at least 13 pg/10⁶ cells. Such mesenchymal stem cells inhibit lymphocyte proliferation.

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate readily into lineages including osteoblasts, myocytes, chondrocytes, and adipocytes (Pittenger, et al., Science, Vol. 284, pg. 143 (1999); Haynesworth, et al., Bone, Vol. 13, pg. 69 (1992); Prockop, Science, Vol. 276, pg. 71 (1997)). In vitro studies have demonstrated the capability of MSCs to differentiate into muscle (Wakitani, et al., Muscle Nerve, Vol. 18, pg. 1417 (1995)), neuronal-like precursors (Woodbury, et al., J. Neurosci. Res., Vol. 69, pg. 908 (2002); Sanchez-Ramos, et al., Exp. Neurol., Vol. 171, pg. 109 (2001)), cardiomyocytes (Toma, et al., Circulation, Vol. 105, pg. 93 (2002); Fakuda, Artif. Organs, Vol. 25, pg. 187 (2001)) and possibly other cell types. In addition, MSCs have been shown to provide effective feeder layers for expansion of hematopoietic stem cells (Eaves, et al., Ann. N.Y. Acad. Sci., Vol. 938, pg. 63 (2001); Wagers, et al., Gene Therapy, Vol. 9, pg. 606 (2002)). Recent studies with a variety of animal models have shown that MSCs may be useful in the repair or regeneration of damaged bone, cartilage, meniscus or myocardial tissues (DeKok, et al., Clin. Oral Implants Res., Vol. 14, pg. 481 (2003)); Wu, et al., Transplantation, Vol. 75, pg. 679 (2003); Noel, et al., Curr. Opin. Investig. Drugs, Vol. 3, pg. 1000 (2002); Ballas, et al., J. Cell. Biochem. Suppl., Vol. 38, pg. 20 (2002); Mackenzie, et al., Blood Cells Mol. Dis., Vol. 27, pgs. 601-604 (2001)). Several investigators have used MSCs with encouraging results for transplantation in animal disease models including osteogenesis imperfecta (Pereira, et al., Proc. Nat. Acad. Sci., Vol. 95, pg. 1142 (1998)), parkinsonism (Schwartz, et al., Hum. Gene Ther., Vol. 10, pg. 2539 (1999)), spinal cord injury (Chopp, et al., Neuroreport, Vol. 11, pg. 3001 (2000); Wu, et al., J. Neurosci. Res., Vol. 72, pg. 393 (2003)) and cardiac disorders (Tomita, et al., Circulation, Vol. 100, pg. 247 (1999). Shake, et al., Ann. Thorac. Surg., Vol. 73, pg. 1919 (2002)). Importantly, promising results also have been reported in clinical trials for osteogenesis imperfecta (Horowitz, et al., Blood, Vol. 97, pg. 1227 (2001); Horowitz, et al. Proc. Nat. Acad. Sci., Vol. 99, pg. 8932 (2002)) and enhanced engraftment of heterologous bone marrow transplants (Frassoni, et al., Int. Society for Cell Therapy, SA006 (abstract) (2002); Koc, et al., J. Clin. Oncol., Vol. 18, pgs. 307-316 (2000)).

In addition, in vitro studies from different laboratories have shown that MSCs can inhibit T-cell proliferation either in mixed lymphocyte cultures or by other stimuli such as antigens and mitogens (Di Nicola, et al., Blood, Vol. 99, pgs. 3638-3843 (2002); Tse, et al., Transplantation, Vol. 75, pgs. 389-397 (2003); Aggarwal, et al., Blood, Vol. 105, pgs. 1815-1822 (2005)). Recent in vitro data demonstrate further that MSCs decrease the secretion of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), and Interferon-γ (IFN-γ), and simultaneously increase production of anti-inflammatory cytokines Interleukin-10 (IL-10) and Interleukin-4 (IL-4) by immune cells. (Aggarwal, 2005). These results indicate that due to immunomodulatory and anti-inflammatory activities, MSCs can be beneficial for treatment of immunological responses which occur in graft-versus-host disease (GVHD), solid organ transplantation, and autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. A clinical case report demonstrating the therapeutic effect of MSCs for acute GVHD supports strongly this hypothesis. (Le Blanc, et al., The Lancet, Vol. 363, pgs. 1439-1441 (2004).)

The TNF-α receptors are expressed on the surface of mesenchymal stem cells. Accumulated data indicate that TNF-α is an important regulator of mesenchymal stem cell function. Incubation of TNF-α with human mesenchymal stem cells in culture upregulates prostaglandin E2 (PGE₂) and keratinocyte growth factor (KGF) secretion, induces indoleamine 2,3 deoxygenase (IDO) enzyme activity and stimulates cell migration. TNF-α has been shown to be present at wound and inflammatory sites, especially in organs targeted by graft-versus-host disease. (Koide, et al., Transplantation, Vol. 64, pgs. 518-524 (1997); Kuroiwa, et al., J. Clin. Invest., Vol. 107, pgs. 1365-1373 (2001); Deans, et al., Exp. Hematol., Vol. 28, pgs. 875-884 (2002); Ellison, et al., J. Clin. Immunol., Vol. 24, pgs. 197-211 (2004)). Thus, such data indicate that expression of TNF-α receptors by mesenchymal stem cells may be critical for immunosuppressive, immunomodulatory, anti-inflammatory, tissue-repairing, or wound-healing activities, as well as migration to sites of inflammation.

There are two types of TNF-α receptors, or TNFRs: Type I (TNFRI), also known as p55, and Type II (TNFRII), also known as p75. (Tartaglia, et al., Proc. Nat. Acad. Sci, Vol. 88, pgs. 9292-9296 (1991).) Both types of TNF-α receptors are present on MSCs; however, TNFRI is the predominant type. (Vancheri, et al., Am. J. Respir. Cell Mol. Biol., Vol. 22, pgs. 628-634 (2000): Debets, et al., Cytokine, Vol. 8, pgs. 80-88 (1996).)

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention now will be described with respect to the drawings wherein:

FIG. 1 is a graph of the correlation between TNFRI expression and the ability of MSCs to inhibit PBMC proliferation in vitro;

FIG. 2 is a graph showing varying TNFRI expression by human mesenchymal stem cells stored at −80° C., −70° C., −60° C., and −50° C.;

FIG. 3 is a graph showing TNFRI expression at varying levels and the correlating ability to inhibit PBMC proliferation in vitro, of human mesenchymal stem cells stored at −80° C. and −50° C.; and

FIG. 4 is a graph showing TNFRI expression by human mesenchymal stem cells stored at −135° C. or below, and then thawed and kept at room temperature for 6, 8, 24, or 32 hours.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is provided a composition comprising mesenchymal stem cells. The mesenchymal stem cells express the TNF-α receptor Type I (TNFR1) in a varying amount effective to inhibit the proliferation of lymphocytes. In one embodiment, the mesenchymal stem cells express TNFRI in an amount of at least 13 pg/10⁶ cells. In another embodiment, the mesenchymal stem cells express TNFRI in an amount, for example, of at least 15 pg/10⁶ cells. In yet another embodiment, the mesenchymal stem cells express TNFRI in an amount of at least 18 pg/10⁶ cells.

Although the scope of the present invention is not to be limited to any theoretical reasoning, Applicants have found that mesenchymal stem cells which express the TNF-α receptor Type I in an amount from, for example, at least 13 pg/10⁶ cells inhibit the proliferation of lymphocytes. Such mesenchymal stem cells are particularly useful in inhibiting immune responses, and more particularly such mesenchymal stem cells are useful in the treatment of graft-versus-host disease; solid organ transplant rejection such as, for example, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, intestine transplant rejection, and kidney transplant rejection; and autoimmune diseases such as, for example, rheumatoid arthritis, multiple sclerosis, Type I diabetes, Crohn's disease, Guillain-Barré syndrome, lupus erythematosus, myasthenia gravis, optic neuritis, psoriasis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, aplastic anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis, Goodpasture's syndrome, Wegener's granulomatosis, coeliac disease, pemphigus, polyarthritis, warm autoimmune hemolytic anemia, and scleroderma.

In one embodiment, the mesenchymal stem cells are obtained from a mammal. The mammal may be a primate, including human and non-human primates.

The mesenchymal stem cells may be a homogeneous composition or may be a mixed cell population enriched in MSCs. Homogeneous mesenchymal stem cell compositions may be obtained by culturing adherent marrow or periosteal cells, and the mesenchymal stem cells may be identified by specific cell surface markers which are identified with unique monoclonal antibodies. A method for obtaining a cell population enriched in mesenchymal stem cells is described, for example, in U.S. Pat. No. 5,486,359. Alternative sources for mesenchymal stem cells include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium.

The varying amount, but at least 13 pg/10⁶ cells, of cellular TNF-α receptor, such as TNF-α receptor Type I, that is expressed in a culture of mesenchymal stem cells may be determined by methods known to those skilled in the art. Such methods include, but are not limited to, quantitative assays such as quantitative ELISA assays, for example. It is to be understood, however, that the scope of the present invention is not to be limited to any particular method for determining the amount of TNF-α receptor and it should be appreciated that the amount of expression can vary.

In one embodiment, the amount of TNF-α receptor expressed by a culture of mesenchymal stem cells is determined by an ELISA assay. In such an assay, a cell lysate from a culture of mesenchymal stem cells is added to a well of an ELISA plate. The well may be coated with an antibody, either a monoclonal or a polyclonal antibody(ies), against the TNF-α receptor. The well then is washed, and then contacted with an antibody, either a monoclonal or a polyclonal antibody(ies), against the TNF-α receptor. The antibody is conjugated to an appropriate enzyme, such as horseradish peroxidase, for example. The well then may be incubated, and then is washed after the incubation period. The wells then are contacted with an appropriate substrate, such as one or more chromogens. Chromogens which may be employed include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate(s) is (are) added, the well is incubated for an appropriate period of time.

Upon completion of the incubation, a “stop” solution is added to the well in order to stop the reaction of the enzyme with the substrate(s). The optical density (OD) of the sample then is measured. The optical density of the sample is correlated to the optical densities of samples containing known amounts of TNF-α receptor in order to determine the amount of TNF-α receptor expressed by the culture of mesenchymal stem cells being tested.

Thus, the present invention provides for the selection of a population of mesenchymal stem cells which express TNF-α receptor Type 1 in an amount, for example, of at least 13 pg/10⁶ cells. However, it should be appreciated by those skilled in the art that the selected population of mesenchymal stem cells of the present invention express the desired amount of TNF-α receptor Type 1 while other populations may not express the desired amount of receptor due to the biological nature of the present invention. It is believed, for example, that approximately 98% of the selected mesenchymal stem cell population of the present invention express the desired TNF-α receptor Type 1 level while the remaining portion of that population may not. Such selected mesenchymal stem cells then may be admixed with an appropriate pharmaceutical carrier for treatment of the diseases and disorders mentioned hereinabove. For example, the mesenchymal stem cells may be administered as a cell suspension including a pharmaceutically acceptable liquid medium for injection.

The mesenchymal stem cells of the present invention are administered to an animal in an amount effective to treat one or more of the above-mentioned diseases or disorders in the animal. The animal may be a mammal, and the mammal may be a primate, including human and non-human primates. The mesenchymal stem cells may be administered systemically, such as, for example, by intravenous, intraarterial, or intraperitoneal administration. The exact dosage of mesenchymal stem cells to be administered is dependent upon a variety of factors, including, but not limited to, the age, weight, and sex of the patient, the disease(s) or disorder(s) being treated, and the extent and severity thereof.

The invention now will be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.

Example 1

In order to investigate the role of TNFRI on the immunosuppressive hMSC activity, hMSCs were transfected transiently by antisense TNFRI type oligonucleotides with the purpose to decrease TNFRI expression (Shen et al., J. Biol. Chem., Vol. 272, pgs. 3550-3553 (1997)). In order to reach different degrees of TNFRI expression inhibition, three different concentrations of oligonucleotides were used for transfection experiments. Non-transfected MSCs and MSCs transfected with a sense oligonucleotide were used as controls. TNFRI expression on hMSCs was analyzed in cell lysates by ELISA, and effect of reduction in TNFRI expression on hMSC capacity to inhibit hPBMC proliferation in vitro was investigated.

Human bone marrow-derived MSCs at Passage 5 from 7 different donors were used for analysis. Cells were obtained from bone marrow aspirates, and isolated using hespan. The cells then were cultured through Passage 5, and frozen in a standard cryopreservation solution containing 5% human serum albumin (HSA) and 10% dimethylsulfoxide in Plasmalyte A. (Baxter) The cells were stored at −80° C. prior to analysis. On the day of the experiment, the hMSCs were thawed, counted, and plated into 6-well tissue culture plates at 2.5×10⁵ cells/well. After overnight incubation, cells were transfected with TNFRI sense or antisense oligonucleotides at concentrations of 1.25, 2.5 and 5 μg/mL according to the transfection reagent manufacturer's protocol (Invitrogen, the Cellfectin transfection reagent product insert). At 24 hours post-transfection, the cells were collected from the plates. One group of cells was lysed, and expression of TNFRI in cell lysates was analyzed by ELISA according to the sTNFRI ELISA protocol (R&D Systems, product insert). TNFRI expression was expressed in pg of receptor per 1×10⁶ cells.

For the ELISA assay, 2.5×10⁵ MSCs per well were lysed directly in wells using 250 μl/well of Cell Lytic-mammalian cell lysis/extraction reagent (Sigma, Catalog No. C-2978) containing a complete protein inhibitor cocktail (Roche). The cell lysates then were centrifuged for 10 minutes at 12,000-14,000 rpm in an Eppendorf centrifuge to remove insoluble material from the lysis buffer solution. The cell lysates then were collected in a new tube for use in the ELISA assay.

An alternative method of cell lysis, i.e., lysis of cell pellets in tubes, also was carried out for frozen cells and for cells collected from tissue culture plates or flasks. Both methods, direct cell lysis in culture plates and lysis of cell pellets in tubes, gave comparable results.

A commercially available ELISA kit, Quantikine®, Human sTNFRI (Catalog No. DRT 100, R&D Systems) was used for the detection of TNFRI in cell lysates. This assay provides for the measurement of both soluble as well as cell-associated TNFRI (Qjwang, et al., Biochemistry, Vol. 36, pg. 6033 (1997).) The assay employs the quantitative sandwich enzyme immunoassay technique. The assay employs a microplate that includes wells that have been pre-coated with a monoclonal antibody specific for TNFRI. TNFRI present in calibrator samples, quality control samples, or samples of MSC cell lysates is captured by the immobilized TNFRI antibody. After washing away any unbound substances, enzyme-linked polyclonal antibodies specific for TNFRI is added to the wells. Following a wash step to remove any unbound enzyme-linked antibody, a substrate solution was added to the wells, and color develops in proportion to the amount of bound TNFRI. The color development then is stopped, and the intensity of the color is measured using an ELISA reader.

The details of the ELISA are given hereinbelow.

50 μl of assay diluent HD1-7, a buffered protein base with preservative, were added to the wells of an ELISA plate. The wells were coated with a monoclonal antibody specific for TNFRI. 200μ1 of either calibrator samples (containing 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.625 pg/ml, or 7.813 pg/ml of soluble human TNFRI), quality control samples (containing 45 pg/ml, 100 pg/ml, or 250 pg/ml of human TNFRI), or cell lysates then were added to the wells. Prior to the addition of the calibration and quality control sample to the wells, such samples were treated with the Cell Lytic-mammalian cell lysis extraction agent (Sigma) and complete protein inhibitor cocktail (Roche) as hereinabove described. The plate then was covered with an adhesive strip, and incubated for 2 hours±10 minutes at room temperature.

The liquid then was decanted from each well by inverting the plate over a sink, and then the plate was washed three times. The plate is washed each time with 400 μl of a wash buffer added to each well. Residual liquid was removed by inverting the plate and blotting.

200 μl of soluble TNFRI polyclonal antibodies conjugated to horseradish peroxidase then were added to each well. The plate then was incubated for 2 hours±10 minutes at room temperature. The liquid then was decanted from each well, and each well was washed three times with 400 μl of wash buffer as hereinabove described.

200 μl of a substrate solution of stabilized hydrogen peroxide and stabilized tetramethylbenzidine chromogen then were added to each well. The plate then was incubated for 20 minutes±10 minutes at room temperature in the dark. 50 μl of a solution of 2N sulfuric acid then were added to each well. The optical density (OD) of each sample then was measured within 30 minutes with a 450 nm test and a 570 nm reference filter. The optical density values then were correlated to the amounts of TNFRI in the cell lysate samples.

Quantitation was achieved by comparing the signal from samples of MSC cell lysates to TNFRI standards assayed at the same time. Each ELISA run provided a calibration curve and included duplicate quality control samples plated in front and after test samples. Quality control samples were used for ELISA run validity assessment. TNFRI expression data were expressed in picograms of receptor per 1×10⁶ cells. The raw data (in pg/ml) reflect TNFRI in picograms per 1×10⁶ cells (2.5×10⁵ cells were lysed in 250 μl of the lysis reagent, thus corresponding to 1×10⁶ cells/nil).

The ELISA values for the calibration samples are given in Table 1 below.

TABLE 1 Calculations for ELISA run calibration standards Back Calculated Theoretical Calculated Mean Concentration OD Concentration Concentration Calibrator of Calibrations OD* Mean Standard for Standards for Standards Sample (pg/mL) Values Value Deviation (pg/mL) (pg/mL) % DFT* % CV* St01 500 2.431 2.437 0.008 498.003 499.923 −0.015 0.3 2.443 501.842 St02 250 1.487 1.476 0.016 252.746 250.306 0.123 1.1 1.464 247.867 St03 125 0.804 0.815 0.015 122.64 124.447 −0.442 1.8 0.825 126.255 St04 62.5 0.453 0.442 0.016 64.774 63.024 0.839 3.5 0.431 61.274 St05 31.25 0.25 0.239 0.016 32.749 30.939 −0.996 6.8 0.227 29.128 St06 15.625 0.143 0.145 0.002 15.765 16.007 2.446 1.5 0.146 16.249 St07 7.813 0.092 0.093 0.001 7.368 7.537 −3.528 1.5 0.094 7.706 *Note: OD—optical density; % DFT—% Difference from Theoretical; CV %—% Coefficient of Variance

The ELISA values for the quality control samples are given in Table 2 below.

TABLE 2 Calculations for ELISA run Quality Control (QC) samples Back Calculated Theoretical Calculated Mean Concentrations OD Concentration Concentration QC for QCs OD* Mean Standard for QCs for QCs Samples: (pg/mL) Values Value Deviation (pg/mL) (pg/mL) % DFT* % CV* Front QCs QC01 45 0.366 0.372 0.008 50.991 51.938 15.417 2.3 0.378 52.884 QCO2 100 0.753 0.733 0.028 113.944 110.572 10.572 3.9 0.713 107.2 QC03 250 1.503 1.509 0.008 256.165 257.454 2.982 0.6 1.515 258.742 Back QCs QC01 45 0.315 0.332 0.024 42.964 45.638 1.418 7.2 0.349 48.312 QC02 100 0.712 0.698 0.021 107.033 104.609 4.609 2.9 0.683 102.185 QC03 250 1.547 1.558 0.015 265.671 267.967 7.187 1 1.568 270.263 *Note: OD—optical density; % DFT—% Difference from Theoretical; CV %—% Coefficient of Variance

Based on the ELISA values for the calibration and quality control samples shown in Tables 1 and 2 hereinabove, TNFRI expression in pg per 1×10⁶ cells for samples of mesenchymal stem cells from the donors was determined. As described hereinabove, the mesenchymal stem cells from each donor were non-transfected, or transfected with a TNFRI sense or antisense oligonucleotide at a concentration of 1.25, 2.5, or 5 μg/ml. The ELISA values and the amount of TNFRI expressed by each of the mesenchymal stem cell samples from each of the donors are given in Table 3 below.

TABLE 3 Calculations for ELISA run test samples TNFRI in OD Calculated Mean pg per hMSC Sample OD* Mean Concentration Concentration 1 × 10⁶ Donor # description: Values Value SD* (pg/mL) (pg/mL) cells % CV* 24 Control (non- 0.385 0.384 0.001 53.989 53.831 53.831 0.4 transfected 0.383 53.674 cells) Control oligo- 0.278 0.266 0.018 37.15 35.186 35.186 6.7 transfected 0.253 33.221 cells 5 ug/mL Control oligo- 0.348 0.352 0.006 48.155 48.785 48.785 1.6 transfected 0.356 49.415 cells 2.5 μg/mL Control oligo- 0.386 0.378 0.012 54.147 52.806 52.806 3.2 transfected 0.369 51.464 cells 1.25 μg/mL TNFRI anti- 0.117 0.113 0.006 11.533 10.79 10.79 5.7 sense oligo- 0.108 10.047 transfected cells 5 μg/mL TNFRI anti- 0.254 0.245 0.013 33.378 31.962 31.962 5.2 sense oligo- 0.236 30.546 transfected cells 2.5 μg/mL TNFRI anti- 0.321 0.311 0.015 43.907 42.257 42.257 4.8 sense oligo- 0.3 40.607 transfected cells 1.25 007 Control (non- 0.368 0.367 0.002 51.306 51.07 51.07 0.6 transfected 0.365 50.833 cells) Control oligo- 0.226 0.219 0.01 28.97 27.866 27.866 4.5 transfected 0.212 26.761 cells 5 μg/mL Control oligo- 0.293 0.272 0.03 39.507 36.128 36.128 11.2 transfected 0.25 32.749 cells 2.5 μg/mL Control oligo- 0.308 0.286 0.032 41.864 38.329 38.329 11.1 transfected 0.263 34.793 cells 1.25 μg/mL TNFRI anti- 0.123 0.114 0.013 12.517 10.949 10.949 11.8 sense oligo- 0.104 9.382 transfected cells 5 μg/mL TNFRI anti- 0.269 0.243 0.037 35.736 31.565 31.565 15.5 sense oligo- 0.216 27.393 transfected cells 2.5 μg/mL TNFRI anti- 0.313 0.303 0.014 42.65 41.078 41.078 4.7 sense oligo- 0.293 39.507 transfected cells 1.25 μg/mL 014 Control (non- 0.377 0.38 0.004 52.726 53.2 53.2 1.1 transfected 0.383 53.674 cells) Control oligo- 0.251 0.249 0.003 32.907 32.592 32.592 1.1 transfected 0.247 32.277 cells 5 μg/mL Control oligo- 0.338 0.315 0.033 46.581 42.887 42.887 10.6 transfected 0.291 39.193 cells 2.5 μg/mL Control oligo- 0.356 0.347 0.013 49.415 47.919 47.919 3.9 transfected 0.337 46.424 cells 1.25 μg/mL TNFRI anti- 0.11 0.104 0.008 10.378 9.379 9.379 8.2 sense oligo- 0.098 8.379 transfected cells 5 μg/mL TNFRI anti- 0.211 0.206 0.008 26.603 25.733 25.733 3.8 sense oligo- 0.2 24.864 transfected cells 2.5 μg/mL TNFRI anti- 0.3 0.294 0.008 40.607 39.664 39.664 2.9 sense oligo- 0.288 38.722 transfected cells 1.25 μg/mL 015 Control (non- 0.475 0.469 0.009 68.284 67.246 67.246 2 transfected 0.462 66.209 cells) Control oligo- 0.278 0.279 0.001 37.15 37.308 37.308 0.5 transfected 0.28 37.465 cells 5 μg/mL Control oligo- 0.34 0.343 0.004 46.896 47.289 47.289 1 transfected 0.345 47.683 cells 2.5 μg/mL Control oligo- 0.419 0.413 0.009 59.37 58.34 58.34 2.2 transfected 0.406 57.31 cells 1.25 μg/mL TNFRI anti- 0.13 0.125 0.007 13.658 12.842 12.842 5.7 sense oligo- 0.12 12.025 transfected cells 5 μg/mL TNFRI anti- 0.253 0.262 0.012 33.221 34.557 34.557 4.6 sense oligo- 0.27 35.893 transfected cells 2.5 μg/mL TNFRI anti- 0.377 0.381 0.005 52.726 53.279 53.279 1.3 sense oligo- 0.384 53.831 transfected cells 1.25 μg/mL 23 Control (non- 0.260 0.255 0.008 40.591 39.632 39.632 3.1 transfected 0.249 38.672 cells) Control oligo- 0.191 0.184 0.010 28.560 27.339 27.339 5.4 transfected 0.177 26.117 cells 5 μg/mL Control oligo- 0.216 0.209 0.009 32.919 31.786 31.786 4.4 transfected 0.203 30.653 cells 2.5 μg/mL Control oligo- 0.222 0.222 0.000 33.965 33.965 33.965 0.0 transfected 0.222 33.965 cells 1.25 μg/mL TNFRI anti- 0.107 0.106 0.001 13.798 13.620 13.620 1.3 sense oligo- 0.105 13.441 transfected cells 5 μg/mL TNFRI anti- 0.206 0.187 0.027 31.176 27.860 27.860 14.4 sense oligo- 0.168 24.544 transfected cells 2.5 μg/mL TNFRI anti- 0.213 0.212 0.001 32.396 32.222 32.222 0.7 sense oligo- 0.211 32.048 transfected cells 1.25 μg/mL 486 Control (non- 0.249 0.249 0.001 41.244 41.148 41.148 0.3 transfected 0.248 41.053 cells) Control oligo- 0.149 0.136 0.018 22.401 19.981 19.981 13.5 transfected 0.123 17.560 cells 5 μg/mL Control oligo- 0.246 0.231 0.022 40.672 37.732 37.732 9.5 transfected 0.215 34.792 cells 2.5 μg/mL Control oligo- 0.263 0.253 0.015 43.915 41.913 41.913 5.9 transfected 0.242 39.911 cells 1.25 μg/mL TNFRI anti- 0.071 0.068 0.004 7.917 7.361 7.361 6.2 sense oligo- 0.065 6.805 transfected cells 5 μg/mL TNFRI anti- 0.142 0.142 0.000 21.096 21.096 21.096 0.0 sense oligo- 0.142 21.096 transfected cells 2.5 μg/mL TNFRI anti- 0.193 0.179 0.021 30.644 27.924 27.924 11.5 sense oligo- 0.164 25.204 transfected cells 1.25 μg/mL 13 Control (non- 0.211 0.209 0.003 34.037 33.659 33.659 1.4 transfected 0.207 33.282 cells) Control oligo- 0.134 0.134 0.01 19.606 19.513 19.513 0.5 transfected 0.133 19.420 cells 5 μg/mL Control oligo- 0.195 0.188 0.011 31.020 29.611 29.611 5.7 transfected 0.180 28.201 cells 2.5 μg/mL Control oligo- 0.207 0.192 0.022 33.282 30.366 38.329 11.4 transfected 0.176 27.451 cells 1.25 μg/mL TNFRI anti- 0.087 0.080 0.010 10.882 9.585 9.585 12.4 sense oligo- 0.073 8.288 transfected cells 5 μg/mL TNFRI anti- 0.156 0.135 0.030 23.708 19.706 19.706 22.6 sense oligo- 0.113 15.703 transfected cells 2.5 μg/mL TNFRI anti- 0.208 0.174 0.048 33.470 27.097 27.097 27.6 sense oligo- 0.140 20.723 transfected cells 1.25 μg/mL *Note: OD—optical density; SD—Standard Deviation; CV %—% Coefficient of Variance

From the above data shown in Table 3, the mean TNFRI expression, in picograms per 1×10⁶ cells, was determined for non-transfected (control) mesenchymal stem cells, as well as mesenchymal stem cells transfected with 1.25, 2.5, or 5 μl/ml of antisense or sense oligonucleotides. The mean TNFRI expression values are given in Table 4 below.

TABLE 4 TNFRI expression by hMSCs transfected with anti-sense and control (sense) oligonucleotides: summary for 7 tested hMSC donors TNFRI expression in pg per 1 × 10⁶ cells Mean hMSC donor #: for 7 486 13 24 007 14 15 23 Donors SD Control (non-  41* 34 54 51 53 67 40 48.57 11.09 transfected cells) TNFRI anti-  7 10 11 11 9 13 14 10.71 2.36 sense oligo- transfected cells 5 μg/mL TNFRI anti- 21 20 32 32 26 35 28 27.71 5.74 sense oligo- transfected cells 2.5 μg/mL TNFRI anti- 28 27 42 41 40 53 32 37.57 9.22 sense oligo- transfected cells1.25 μg/mL Control (sense) 20 20 35 28 33 37 27 28.57 6.85 oligo-transfected cells 5 μg/mL Control (sense) 38 30 49 36 43 47 32 39.29 7.30 oligo-transfected cells 2.5 μg/mL Control (sense) 42 30 53 38 48 58 34 43.29 10.21 oligo-transfected cells 1.25 μg/mL *Note: These values represent mean TNFRI numbers (from table 3, column 8: “TNFRI in pg per 1 × 10⁶ cells”) rounded to whole numbers

A second group of transfected cells was used for investigation of the effect of hMSCs on hPBMC proliferation in vitro. Human PBMCs from two different donors were used for this assay. PBMCs were isolated from leukopheresed blood using Ficoll-Paque gradient centrifugation according to the manufacturer's protocol (Amersham Biosciences, Ficoll-Paque Plus product insert). Cells were stored frozen at −80° C. in a medium including 90% FBS and 10% DMSO prior to analysis. On the day of the experiment hPBMCs were thawed, counted and plated into 96-well tissue culture plates at 1×10⁵ cells/well together with hMSCs (1×10⁴ cells/well). A combination of anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies was used to stimulate lymphocyte proliferation that represents an in vitro model for immune cell activation characteristics of GVHD and rejection of allogeneic organs. (Trickett, et al., J. Immunol. Methods, Vol. 275, pgs. 251-255 (2003); Koulova, et al., J. Exp. Med., Vol. 173, No. 3, pgs. 759-762 (1991); Foster, et al., Transplantation, Vol. 76, No. 6; Czitrom, Clin. Ortho. Relat. Res., Vol. 326, pgs. 11-24 (1996)). The plates then were incubated in a humidified atmosphere containing 5% CO₂. The proliferation of PBMCs alone and in the presence of MSCs was measured at day 5 from culture initiation by the addition of [Methyl-³H]-thymidine at 1 μCi/well for the final 18-20 hrs of culture. After labeling, the cells were transferred onto a glass filter using a 96-well plate harvester, and radioactivity incorporated into DNA was measured by a liquid scintillation beta-counter. The uptake of [Methyl-³H]-thymidine into DNA in counts per minute (cpm) represents hPBMC proliferation. Final results were expressed as % inhibition of PBMC proliferation in the presence of MSCs calculated as:

100%−[Proliferation(PBMC+MSC,cpm)×100/Proliferation(PBMC,cpm)]

The results for the mesenchymal stem cells from each of the donors are given in Table 5 below.

TABLE 5 Inhibition of CD3/CD28-induced hPBMC proliferation by hMSCs transfected with anti-sense and control (sense) oligonucleotides: summary for 7 tested hMSC donors % inhibition of hPBMC proliferation by hMSCs hMSC donor #: 486 13 24 007 14 15 23 Mean % hPBMC donor #: for 7 2 3 2 3 3 3 3 2 2 3 donors SD Control (non- 65 73 82 94 70 66 82 62 68 91 75.30 11.26 transfected cells) TNFRI anti-sense 40 45 46 68 32 10 39 19 38 52 38.90 16.29 oligo-transfected cells 5 μg/mL TNFRI anti-sense 83 90 59 86 ND 73 ND 63 47 58 69.88 15.48 oligo-transfected cells 2.5 μg/mL TNFRI anti-sense 62 74 86 ND 72 64 57 ND 72 80 70.88 9.58 oligo-transfected cells1.25 μg/mL Control (sense) 38 87 60 77 58 77 62 44 52 53 60.80 15.50 oligo-transfected cells 5 μg/mL Control (sense) 60 91 67 ND ND 62 66 57 70 95 71.00 14.22 oligo-transfected cells 2.5 μg/mL Control (sense) 87 ND 68 71 66 68 36 ND 49 85 70.57 12.77 oligo-transfected cells 1.25 μg/mL Note: ND—no data

The above data with respect to inhibition of CD3/CD28 induced PBMC proliferation were correlated to the mean TNFRI expression data shown in Table 4 hereinabove. The correlated data with respect to mean TNFRI expression and inhibition of CD3/CD28 induced PBMC proliferation are given in Table 6 below.

TABLE 6 TNFRI expression and effect on hPBMC proliferation in vitro by hMSCs transfected with TNFRI oligonucleotides Oligonucleo- % Inhibition of TNFRI tide concen- hPBMC Expression in Human MSCs tration proliferation pg/1 × 10⁶ MSCs condition (pg/mL) (Mean ± SD) (Mean ± SD) Untransfected Not appli- 75.30 ± 11.26 48.57 ± 11.09 (Control MSCs) cable Antisense oligo- 1.25 70.88 ± 9.58  37.57 ± 9.22  nucleotide 2.5 69.88 ± 15.48 27.71 ± 5.74  5 38.90 ± 16.29 10.71 ± 2.36  Sense oligonucleo- 1.25 70.57 ± 12.77 43.29 ± 10.21 tide (control 2.5 71.00 ± 14.22 39.29 ± 7.30  oligonucleotide) 5 60.80 ± 15.50 28.57 ± 6.85 

The results from these experiments show that hMSCs with decreased expression of TNFR type I (TNFRI) lose their ability to suppress hPBMC proliferation in vitro. The data support the premise that the expression of TNFRI is an essential link to the suppression of PBMC proliferation by MSCs. Thus, TNFRI can be used as a potency marker for MSC immunomodulative activity. Based on the obtained data, a potency threshold of 13.07 pg of, for example, TNFRI (mean±SD) per 1×10⁶ cells correlates with less than 50% inhibition of hPBMC proliferation (Table 6, FIG. 1). Thus, the present invention illustrates that those non-potent MSCs of a selected MSC population of at least one embodiment of the present invention are those cells expressing less than 13 pg TNFRI per 1×10⁶ cells.

Example 2 TNFRI is a Temperature-Sensitive Marker of hMSC Functionality

Ex vivo handling of mammalian cells is restricted by a number of factors including temperature. For example, low temperatures such as −80±5° C., or lower, even as low as −135° C. or below (liquid nitrogen) are required for cell storage whereas ex vivo cell expansion requires a temperature of 37±0.5° C. Cell exposure to temperatures outside of the optimal ranges may lead to a decrease in cell functionality or cell death. Mammalian cells are able to withstand short-term minor temperature fluctuations; however, each type of cells has its own temperature tolerance range for cell culture maintenance, shipping, and storage.

As discussed herein, it is believed that the expression level of TNFRI on some hMSCs of the selected population of the present invention correlates with hMSC immunosuppressive activity. For example, the level of TNFRI expression by some of those hMSCs of less than 13 pg/10⁶ cells has been determined as a threshold, below which hMSCs begin to lose their ability to suppress an immune response (See FIG. 1). Thus, TNFRI expression is a marker of hMSC immunosuppression, an activity that is believed essential for MSCs to be efficacious for treatment of immunological reactions taking place in GVHD, organ rejection, autoimmune diseases, and other diseases. Here, effects of temperature fluctuations during storage of frozen hMSCs as well as the effect of time of exposure of cells to room temperature on expression of TNFRI on hMSCs was investigated.

Effect of Store Temperature Fluctuations on TNFRI Expression and hMSC Immunosuppressive Potential.

The objective of these experiments was to investigate the ability of the selected hMSCs to retain their functional characteristics after an exposure to temperatures above −80° C., which are not optimal temperatures for storage of frozen cells. Human MSCs were frozen at passage 5 and placed for storage in a freezer at −80±5° C. After several weeks, bags of frozen cells were removed from the −80±5° C. freezer and placed at either −70±5° C., −60±5° C., or −50±5° C. for 72±2 hours. After 72±2 hours, the bags were returned to storage at −80±5° C. for at least 24 hours before thaw and analysis. A set of bags moved from one −80±5° C. freezer to another, following the same schedule as the other bags, served as a control. On the day of the experiment the bags containing the cells were thawed, cells were counted, and cell lysates for the TNFRI ELISA were prepared as described in Example 1. The TNFRI ELISA was performed as described in Example 1. Results are summarized in FIG. 2 (bars show mean TNFRI values±SD for 3 hMSC bags). The data showed that exposure of hMSCs to temperatures of −60±5° C. or −50±5° C. decreases the TNFRI expression level: the level of TNFRI detected by ELISA was below the determined hMSC potency threshold of 13 pg/10⁶ cells (represented by the solid line on the graph).

Parallel with TNFRI measurement, two bags with hMSCs stored at −80±5° C. (optimal storage temperature served as a control) and at −50±5° C. (corresponding to a +30° C. greater than the −80±5° C. optimal storage temperature) were used for investigation of hMSC immunosuppressive activity. The ability of the MSCs to suppress anti-CD3/CD28-induced proliferation of hPBMCs in vitro was evaluated as described in Example 1. The results showed that hMSCs stored at −50±5° C. lost their ability to suppress hPBMC proliferation, whereas cells stored at −80±5° C. inhibited hPBMC proliferation by 92%, but not up to 100% (FIG. 3, dark bars represent mean±SD % inhibition of hPBMC proliferation. Numbers inside the dark bars show numerical values). The immunosuppressive activity of MSCs is dependent on the level of TNFRI expression: for example, some cells expressing more than 13 pg/10⁶ cells of TNFRI, which was determined as an MSC immunosuppressive potential threshold, are biologically active, while others cells with the TNFRI level below 13 pg/10⁶ cells are not (FIG. 3, light bars represent mean±SD of the TNFRI expression level. Numbers inside the light bars show numerical values). Thus, non-optimal storage temperatures decrease TNFRI expression on hMSCs, and which correlates with decrease in hMSC functionality.

Effect of Cell Exposure Time to Room Temperature on TNFRI Expression on hMSC.

The results of this experiment serve as additional evidence that TNFRI expression on hMSCs is decreasing under cell exposure to non-optimal temperatures. In this experiment the effect of cell suspension storage at room temperature on TNFRI expression was studied. Two hMSC lots were used in the experiment. Bags containing hMSCs were stored at ≦−135° C. prior to the experiment. On the day of the experiment the cells were thawed and diluted with Plasmalyte A physiological solution (Baxter) in a manner that mimics the current cell processing for intravenous hMSC administration at clinical sites. The thawed and diluted hMSCs were kept at room temperature (22° C.-24° C.), and samples were taken and tested for the amount of TNFRI at 0 (immediately post-thaw—baseline), 6, 8, 10, 24, and 32 hours post-thawing. The results showed that exposure of hMSCs to room temperature decreased the TNFRI expression level on those hMSCs (FIG. 4, bars represent mean±SD of the TNFRI expression level for 2 hMSC lots. The solid line represents the TNFRI expression level of 13 pg/10⁶ cells, which is the hMSC potency threshold). The significant decrease in TNFRI expression was observed at 24 hours and 32 hours, and it correlated with a significant decrease in cell viability (below 20%, data not shown).

Thus, the experiments described above show that TNFRI expression by hMSCs is sensitive to temperature, and TNFRI can be used as a marker of functionality of hMSC that were exposed to non-optimal temperatures during storage, shipping or cell processing.

The disclosures of all patents, publications, including published patent applications, depository accession numbers, and database accession numbers are hereby incorporated by reference to the same extent as if each patent, publication, depository accession number, and database accession number were specifically and individually incorporated by reference.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims. 

1. Mesenchymal stem cells which express TNF-α receptor Type I in an amount of at least 13 pg/10⁶ cells produced by a method comprising: i) obtaining at least one cell population of mesenchymal stem cells from at least one donor; ii) determining the amount of TNF-α receptor Type I expressed by the mesenchymal stem cells in each of said at least one cell population(s); and iii) selecting mesenchymal stem cells which express TNF-α receptor Type I in an amount of at least 13 pg/10⁶ cells.
 2. The mesenchymal stem cells of claim 1, wherein said selected mesenchymal stem cells express TNF-α receptor Type I in an amount of at least 15 pg/10⁶ cells.
 3. The mesenchymal stem cells of claim 1, wherein said selected mesenchymal stem cells express TNF-α receptor Type I in an amount of at least 18 pg/10⁶ cells.
 4. The mesenchymal stem cells of claim 1, wherein said mesenchymal stem cells are human mesenchymal stem cells.
 5. The mesenchymal stem cells of claim 1, wherein the mesenchymal stem cells are obtained from a single donor.
 6. The mesenchymal stem cells of claim 1, where the cells are cultured for at least five passages.
 7. The mesenchymal stem cells of claim 1, wherein the method further comprises cryopreserving the mesenchymal stem cells.
 8. The mesenchymal stem cells of claim 7, wherein the mesenchymal stem cells are cryopreserved in a solution comprising 5% human serum albumin (HAS) and 10% dimethylsulfoxide (DMSO).
 9. The mesenchymal stem cells of claim 8, wherein the cryopreservation solution further comprises Plasmalyte A.
 10. The mesenchymal stem cells of claim 1, wherein said mesenchymal stem cells have been subjected to cryopreservation at a temperature of about −80° C.
 11. A composition enriched in non-transfected mesenchymal stem cells which express TNF-α receptor Type I in an amount of at least 13 pg/10⁶ cells and wherein the mesenchymal stem cells suppress hPBMC proliferation.
 12. The composition of claim 11, wherein the mesnechymal stem cells express TNF-α receptor Type I in an amount of at least 15 pg/10⁶ cells.
 13. The composition of claim 11, wherein the mesnechymal stem cells express TNF-α receptor Type I in an amount of at least 18 pg/10⁶ cells.
 14. The composition of claim 11, wherein the mesenchymal stem cells are cryopreserved.
 18. The composition of claim 14, wherein the composition further comprises a solution comprising 5% human serum albumin (HAS) and 10% di methylsulfoxide.
 19. The composition of claim 18, wherein the cryopreservation solution further comprises Plasmalyte A.
 20. A composition comprising culturally expanded mesenchymal stem cells which express TNF-α receptor Type I in an amount of at least 13 pg/10⁶ cells and wherein the mesenchymal stem cells suppress hPBMC proliferation. 